Hematopoietic stem cell transplantation (HSCT) is a life-saving therapy for a variety of hematological disorders. When using allogeneic HSCT as a therapy, however, the graft-versus-tumor (GVT) effect is in large part mediated by the graft-versus-host reaction (GVHR), which also often leads to graft-versus-host disease (GVHD). Therefore, controlling alloreactivity to prevent GVHD while preserving GVT effects represents a true dilemma and the key challenge in HSCT. The long-term goal of this project is to exploit the ability of inducible T regulatory cells (Tregs) to contain alloreactive T cells in a desirable manner, and thus to prevent GVHD while retaining GVT effects. The central hypothesis is that forced Foxp3 expression will convert a T-cell clone into Tregs with a known TCR specificity, which can be activated by a specific antigen. Because the onset and duration of activation can be regulated in antigen-specific T cells, the suppressive function of these Tregs can be controlled at will. Thereby, this strategy is expected to have the potential to control GVHD while preserving GVT effects. Three specific aims are designed to test the central hypothesis: 1) Control GVHD by Foxp3-transduced, CD4+CD25- TCR transgenic (Tg) cells. CD4+CD25- cells will be isolated from WT or TCR Tg mice and converted into Tregs by Foxp3-transduction via retroviral infection. The effects of WT or TCR Tg Tregs will be tested in the prevention of GVHD after activation with recipient alloantigen. 2) Control GVHD by a Foxp3-transduced, CD4+CD25- T-cell clone. An antigen-specific T-cell clone will be transduced with Foxp3 and used as Tregs. The effects of Tregs will be tested in the prevention and treatment of GVHD after activation by recipient alloantigen or a peptide antigen. 3) Maintain GVT activity with inducible Tregs activated with peptide antigen for a short period of time. In a GVHD plus tumor model, the ability of Tregs to control GVHD while maintaining GVT effects will be compared under two conditions where Tregs are constantly activated by recipient alloantigen or transiently activated by a peptide antigen.